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1.
Cell ; 138(3): 463-75, 2009 Aug 07.
Article in English | MEDLINE | ID: mdl-19665970

ABSTRACT

Telomeres are thought to be maintained by the preferential recruitment of telomerase to the shortest telomeres. The extension of the G-rich telomeric strand by telomerase is also believed to be coordinated with the complementary synthesis of the C strand by the conventional replication machinery. However, we show that under telomere length-maintenance conditions in cancer cells, human telomerase extends most chromosome ends during each S phase and is not preferentially recruited to the shortest telomeres. Telomerase rapidly extends the G-rich strand following telomere replication but fill-in of the C strand is delayed into late S phase. This late C-strand fill-in is not executed by conventional Okazaki fragment synthesis but by a mechanism using a series of small incremental steps. These findings highlight differences between telomerase actions during steady state versus nonequilibrium conditions and reveal steps in the human telomere maintenance pathway that may provide additional targets for the development of anti-telomerase therapeutics.


Subject(s)
Telomerase/metabolism , Telomere/metabolism , Cell Cycle , Cell Line, Tumor , HeLa Cells , Humans , S Phase , Saccharomyces cerevisiae/enzymology
2.
EMBO Rep ; 7(2): 225-30, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16374507

ABSTRACT

A central function of telomeres is to prevent chromosome ends from being recognized as DNA double-strand breaks (DSBs). Several proteins involved in processing DSBs associate with telomeres, but the roles of these factors at telomeres are largely unknown. To investigate whether the Mre11/Rad50/Nbs1 (MRN) complex is involved in the generation of proper 3' G-overhangs at human telomere ends, we used RNA interference to decrease expression of MRN and analysed their effects. Reduction of MRN resulted in a transient shortening of G-overhang length in telomerase-positive cells. The terminal nucleotides of both C- and G-rich strands remain unaltered in Mre11-diminished cells, indicating that MRN is not responsible for specifying the final end-processing event. The reduction in overhang length was not seen in telomerase-negative cells, but was observed after the expression of exogenous telomerase, which suggested that the MRN complex might be involved in the recruitment or action of telomerase.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Repair Enzymes/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Telomere/metabolism , Acid Anhydride Hydrolases , Base Sequence , Cell Cycle Proteins/genetics , DNA Repair Enzymes/genetics , DNA-Binding Proteins/genetics , HeLa Cells , Humans , MRE11 Homologue Protein , Nuclear Proteins/genetics , Polymerase Chain Reaction , RNA Interference , RNA, Small Interfering/metabolism , Telomere/chemistry
3.
Cell Cycle ; 4(11): 1467-70, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16258279

ABSTRACT

Telomeres protect chromosomes from degradation and loss of vital sequence, block end-end fusion, and allow the cell to distinguish between broken ends and chromosome ends. Mammalian telomeres end in single-stranded (TTAGGG)-rich 3'-overhangs that are tucked back into the preceding double stranded region to form a T-loop. The end structure of mammalian telomeres has just started to be elucidated and through this extra views we highlight one aspect of that structure. We have recently identified the terminal nucleotides of both the C-rich and G-rich telomere strands in human cells and showed that approximately 80% of the C-rich strands terminate precisely in ATC-5', while the last base of the G-strand is less precise. This finding has important implications for the processing events that act on the telomere ends post-replication. While the mechanism behind this phenotype is yet to be unraveled, we discuss potential models that could explain the last base specificity.


Subject(s)
Base Sequence/genetics , Chromosomes, Human/chemistry , Telomere/chemistry , Chromosomes, Human/genetics , Cytosine/chemistry , Guanine/chemistry , Humans , Telomere/genetics
4.
EMBO J ; 24(14): 2667-78, 2005 Jul 20.
Article in English | MEDLINE | ID: mdl-15973431

ABSTRACT

The hallmarks of telomere dysfunction in mammals are reduced telomeric 3' overhangs, telomere fusions, and cell cycle arrest due to a DNA damage response. Here, we report on the phenotypes of RNAi-mediated inhibition of POT1, the single-stranded telomeric DNA-binding protein. A 10-fold reduction in POT1 protein in tumor cells induced neither telomere fusions nor cell cycle arrest. However, the 3' overhang DNA was reduced and all telomeres elicited a transient DNA damage response in G1, indicating that extensive telomere damage can occur without cell cycle arrest or telomere fusions. RNAi to POT1 also revealed its role in generating the correct sequence at chromosome ends. The recessed 5' end of the telomere, which normally ends on the sequence ATC-5', was changed to a random position within the AATCCC repeat. Thus, POT1 determines the structure of the 3' and 5' ends of human chromosomes, and its inhibition generates a novel combination of telomere dysfunction phenotypes in which chromosome ends behave transiently as sites of DNA damage, yet remain protected from nonhomologous end-joining.


Subject(s)
Chromosomes, Human/metabolism , DNA Damage/physiology , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Alternative Splicing , Cell Proliferation , Chromosomes, Human/chemistry , HeLa Cells , Humans , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Shelterin Complex , Telomere/chemistry , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/genetics
5.
Mol Cell ; 18(1): 131-8, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15808515

ABSTRACT

Mammalian telomeres end in single-stranded, G-rich 3' overhangs resulting from both the "end-replication problem" (the inability of DNA polymerase to replicate the very end of the telomeres) and postreplication processing. Telomeric G-rich overhangs are precisely defined in ciliates; the length and the terminal nucleotides are fixed. Human telomeres have very long overhangs that are heterogeneous in size (35-600 nt), indicating that their processing must differ in some respects from model organisms. We developed telomere-end ligation protocols that allowed us to identify the terminal nucleotides of both the C-rich and the G-rich telomere strands. Up to approximately 80% of the C-rich strands terminate in CCAATC-5', suggesting that after replication a nuclease with high specificity or constrained action acts on the C strand. In contrast, the G-terminal nucleotide was less precise than Tetrahymena and Euplotes but still had a bias that changed as a function of telomerase expression.


Subject(s)
Chromosomes, Human/genetics , Chromosomes, Human/metabolism , Telomere/genetics , Telomere/metabolism , Animals , Base Sequence , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Euplotes/genetics , Humans , Telomerase/metabolism , Tetrahymena/genetics
6.
J Cell Sci ; 116(Pt 17): 3531-41, 2003 Sep 01.
Article in English | MEDLINE | ID: mdl-12893812

ABSTRACT

The relationship between gap junctional intercellular communication (GJIC) and mammary cell (CID-9) differentiation in vitro was explored. CID-9 cells differentiate and express beta-casein in an extracellular matrix (ECM)- and hormone-dependent manner. In response to interaction with the ECM, cells in culture modulated the expression of their gap junction proteins at the transcriptional and post-translational levels. In the presence of EHS-matrix, connexins (Cx)26, 32 and 43 localized predominantly to the plasma membrane, and enhanced GJIC [as measured by Lucifer Yellow (LY) dye transfer assays] was noted. Inhibition of GJIC of cells on EHS-matrix with 18 alpha glycyrrhetinic acid (GA) resulted in reversible downregulation of beta-casein expression. In the presence of cAMP, cells cultured on plastic expressed beta-casein, upregulated Cx43 and Cx26 protein levels and enhanced GJIC. This was reversed in the presence of 18 alpha GA. cAMP-treated cells plated either on a non-adhesive PolyHEMA substratum or on plastic supplemented with function-blocking anti-beta 1 integrin antibodies, maintained beta-casein expression. These studies suggest that cell-ECM interaction alone may induce differentiation through changes in cAMP levels and formation of functional gap junctions. That these events are downstream of ECM signalling was underscored by the fact that enhanced GJIC induced partial differentiation in mammary epithelial cells in the absence of an exogenously provided basement membrane and in a beta 1-integrin- and adhesion-independent manner.


Subject(s)
Cell Communication/physiology , Cell Differentiation/physiology , Cell Membrane/metabolism , Extracellular Matrix/metabolism , Gap Junctions/metabolism , Glycyrrhetinic Acid/analogs & derivatives , Animals , Antibodies, Blocking/pharmacology , Caseins/metabolism , Cell Communication/drug effects , Cells, Cultured , Connexin 26 , Connexin 43/metabolism , Connexins/metabolism , Cyclic AMP/metabolism , Female , Gap Junctions/drug effects , Glycyrrhetinic Acid/pharmacology , Immunohistochemistry , Integrin beta1/drug effects , Integrin beta1/metabolism , Isoquinolines/metabolism , Mice , Polyhydroxyethyl Methacrylate , Gap Junction beta-1 Protein
7.
J Biol Chem ; 278(22): 19826-33, 2003 May 30.
Article in English | MEDLINE | ID: mdl-12654912

ABSTRACT

Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.


Subject(s)
Carrier Proteins/metabolism , Endocytosis , Fungal Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligase Complexes , Ubiquitin/metabolism , Vesicular Transport Proteins , Amino Acid Sequence , Base Sequence , Carrier Proteins/chemistry , DNA Primers , Endosomal Sorting Complexes Required for Transport , Fungal Proteins/chemistry , Guanine Nucleotide Exchange Factors , Molecular Sequence Data , Protein Binding , Saccharomyces cerevisiae Proteins/chemistry , Sequence Homology, Amino Acid
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